Association of a placental Interleukin-6 genetic variant ... … · cluding cases of spontaneous premature preterm rupture of the membranes, placental abruption, fetal vascular malperfusion,

RESEARCH ARTICLE Open Access

Association of a placental Interleukin-6genetic variant (rs1800796) with DNAmethylation, gene expression and risk ofacute chorioamnionitisChaini Konwar1,2, Giulia F. Del Gobbo1,2, Jefferson Terry1,3 and Wendy P. Robinson1,2*

Abstract

Background: Acute chorioamnionitis (aCA), inflammation of the placenta and fetal membranes, is a frequentlyreported lesion in preterm deliveries. Genetic variants in innate immune system genes such as Interleukin-6 (IL6)may contribute to the placenta’s inflammatory response, thus predisposing some pregnancies to aCA. Thesegenetic variants may modulate molecular processes such as DNA methylation and gene expression, and in turnmight affect susceptibility to aCA. Currently, there is remarkably little research on the role of fetal (placental) geneticvariation in aCA. We aimed to explore the associations between genetic variants in candidate immune-systemgenes and susceptibility towards inflammatory responses in the placenta, which is linked to a strong inflammatoryresponse in the newborn.

Methods: DNA samples from 269 placentas (72 aCA cases, 197 non-aCA cases) were collected for this study.Samples were genotyped at 55 ancestry informative markers (AIMs) and 16 additional single nucleotidepolymorphisms (SNPs) in 12 candidate innate immune system genes using the Sequenom iPLEX Gold Assay.Publicly available datasets were used to obtain DNA methylation (GSE100197, GSE74738, GSE115508, GSE44667,GSE98224) and gene expression data (GSE44711, GSE98224).

Results: Differences in IL6 placental allele frequencies were associated with aCA (rs1800796, p = 0.04) with the CCgenotype specifically implicated (OR = 3.1; p = 0.02). In a subset of the placental samples (n = 67; chorionic villi), weshowed that the IL6 SNP (rs1800796) was associated with differential DNA methylation in five IL6-related CpG sites(cg01770232, cg02335517, cg07998387, cg13104385, and cg0526589), where individuals with a CC genotypeshowed higher DNA methylation levels than individuals carrying the GG genotype. Using two publicly availabledatasets, we observed that the DNA methylation levels at cg01770232 negatively correlated with IL6 geneexpression in the placenta (r = − 0.67, p < 0.004; r = − 0.56, p < 2.937e-05).

Conclusions: We demonstrated that the minor C allele at the IL6 SNP (rs1800796), which is largely limited to EastAsian populations, is associated with the presence of aCA. This SNP was associated with increased DNA methylationat a nearby MEPC2 binding site, which was also associated with decreased expression of IL6 in the placenta.Decreased expression of IL6 may increase vulnerability to microbial infection. Additional studies are required toconfirm this association in Asian populations with larger sample sizes.

Keywords: Placenta, Chorioamnionitis, SNP, Interleukin-6, DNA methylation, Gene expression

* Correspondence: [email protected] Children’s Hospital Research Institute, 950 W 28th Ave, Vancouver, BCV5Z 4H4, Canada2Department of Medical Genetics, University of British Columbia, Vancouver,BC V6H 3N1, CanadaFull list of author information is available at the end of the article

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Konwar et al. BMC Medical Genetics (2019) 20:36 https://doi.org/10.1186/s12881-019-0768-0

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BackgroundPreterm birth (PTB) refers to all births occurring before37 weeks of gestation, as defined by the World HealthOrganization [1]. PTB occurs in approximately 11% oflive births worldwide, although there is substantial vari-ability in rates on a per-country basis [2]. Children whoare born preterm are at a higher risk for life-long healthcomplications, including chronic lung disease, cerebralpalsy, mental retardation, attention-deficit hyperactivitydisorder, and learning disabilities [3, 4]. Clinically, PTBcan be categorized into spontaneous PTB (sPTB) ormedically-indicated PTB (iatrogenic) [1]. sPTB, makingup the majority of PTBs, typically results from a dysreg-ulation of inflammatory signalling pathways [5]. Thisoften presents as acute chorioamnionitis (aCA), which ischaracterized by an infiltration of maternal neutrophilsinto the chorioamniotic membranes, typically in re-sponse to an ascending microbial infection from thegenital tract. This acute placental inflammation can alsobe triggered by non-microbial “danger signals” includingcellular stress and/or cell death [6, 7].Genetic susceptibility for aCA can be hypothesized

based on: i) high heritability estimates of PTB (15–30%)[8, 9], ii) evidence supporting familial segregation ofPTB [10, 11], (iii) association of placental histopatho-logical inflammatory lesions with recurrent PTB [12, 13],and (iv) ethnic disparities in PTB [14–16] and chorioam-nionitis rates [17]. Inherited differences in immunesystem genes also influence susceptibility to microbialinfection [18–21], which is a well-known cause of aCA.In addition, a strong genetic predisposition underliesmany infectious and inflammatory diseases, particularlyin early childhood [22–24]; this may also hold true for inutero susceptibility for aCA.Studies investigating candidate genes have reported

that maternal and fetal genetic variation in Toll-like re-ceptors (TLRs) is associated with sPTB [25–27]. Allelicvariation in TLR genes has been shown to modulateimmune responses during parturition, and thus conferan altered risk of preterm delivery [28]. Genetic variantsin cytokine genes such as Interleukin-6 (IL6) have alsobeen associated with intrauterine infection and/or in-flammation in sPTB [29–31]. Furthermore, elevated con-centration of IL6 in maternal serum, cervical secretionsand amniotic fluid are associated with sPTB [32–35].Recently, a genome-wide association study investigating> 40,000 women of European ancestry identified severalgenetic variants associated with sPTB [36]. Althoughvariants in EBF1, EEFSEC, and AGTR2 genes were repli-cated in an independent cohort of > 8000 women, noneof the identified genes had been previously identified insPTB or known to have a direct role in inflammatorymechanisms [36]. While these studies have providedsome insight on genetic variation linked to sPTB, rarely

are the same loci reported with sPTB risk. sPTB isheterogeneous in etiology [37], thus inconsistent pheno-typing of sPTB cases and differences in population struc-ture may explain the discrepancies across these studies.Genetic variants within coding regions may directly

affect protein function, while those in regulatory regionsmay affect molecular processes such as DNA methyla-tion [38–40] that are involved in regulating gene expres-sion [41, 42]. Alternatively, genetic variants can alter thebinding site of transcription factors and affect geneexpression, which then influences DNA methylationlevels, suggesting DNA methylation as a consequence ofgene regulation [43]. Irrespective of the underlyingmechanism, these effects can in turn affect susceptibilityto inflammatory diseases. For example in rheumatoidarthritis, DNA methylation at an IL6-related CpG wasaltered in affected patients, and a negative relationshipbetween DNA methylation and IL6 mRNA levels wasobserved, suggesting a DNA methylation-dependentregulation of IL6 transcription [44]. While increasedserum levels of IL6 have been previously reported inaCA [45–47], these studies did not take into account thegenotype at the IL6 locus and/or the DNA methylationstatus of the IL6-related CpGs. Furthermore, the mater-nal genotype is often investigated although the placentalgenotype may be more relevant in terms of mediatingpregnancy-related inflammation. Elucidating these com-plex relationships between genotype, DNA methylationand gene expression is important to improve our under-standing of the genetic regulation of placentalinflammation.In this study, we investigated the association between

16 candidate SNPs in 12 innate immune system genesand the presence of aCA. These SNPs were chosenbased on published reports of an association with chor-ioamnionitis [48–50], placental inflammation [51, 52] orneonatal sepsis/infection [53–55]. We validated theseassociations in a population of 269 placentas, of which72 were affected with aCA and 197 were unaffected(non-aCA). Further, we investigated whether aCA-asso-ciated SNPs showed also a correlation with altered DNAmethylation of the associated gene, and determinedwhether DNA methylation levels correlated with geneexpression.

MethodsStudy cohortEthics approval was obtained from the University ofBritish Columbia Children’s & Women’s Research EthicsBoard (H04–70488). The study cohort is based on anongoing collection of samples for our Epigenetics inPregnancy study (EPIC), and overlaps with samplesdescribed in previous publications related to placentalDNA methylation [56–58]. Most cases were obtained in

Konwar et al. BMC Medical Genetics (2019) 20:36 Page 2 of 12

a deidentified manner with pathology and birth informa-tion available, but limited information on maternalhealth or demographics.Placentas were collected from pregnancies delivered at

the Children’s & Women’s Health Centre, Vancouver,Canada. Placentas from 72 aCA cases were selectedbased on a diagnosis of aCA determined by pathologicalexamination of the placenta and associated membranesusing consensus histological criteria [59]. Another set of197 non-aCA cases were identified from this samecollection of placentas. These consisted of 73 PTBs withno evidence of aCA and/or placental inflammation in-cluding cases of spontaneous premature preterm ruptureof the membranes, placental abruption, fetal vascularmalperfusion, acute hypoxic ischemic event, and pretermlabor), in addition to 124 term (> 37 weeks gestation)cases from healthy, uncomplicated pregnancies. Criteriafor exclusion were fetal and/or placental chromosomalabnormalities, fetal malformations, congenital abnormal-ities, intrauterine growth restriction [60], preeclampsia(PE) [61], and hypertension.Demographic characteristics of the study cohort is

presented in Table 1. Of the variables investigated gesta-tional age (GA) at delivery was significantly differentbetween the aCA cases and non-aCA cases in our studycohort as aCA cases are associated with PTB. Althoughmale fetuses are reported to be at an increased risk ofadverse pregnancy outcomes including chronic inflam-mation, neonatal sepsis, and stillbirth; in our study, fetalsex was not significantly different between aCA andnon-aCA groups.Further, the aCA cases and the non-aCA cases were

sampled from a single urban population (Vancouver)and delivered at a single centre, BC Children’s &Women’s Health Centre which is located in a highsocio-economic status neighborhood. Of the docu-mented observations for maternal smoking status (80/269), almost all (79/80) identified themselves asnon-smokers. Additionally, the most reproducible

finding between maternal smoking and altered DNAmethylation is observed at sites linked with AHRR andCYP1A1. We therefore tested for differences at thesesites and did not observe altered DNA methylationassociated with our pathology at these sites in our studycohort (data not shown).Chorionic villus samples were obtained from the fetal

side of the placenta at multiple distinct sites (cotyle-dons). Samples were thoroughly washed with phosphate-buffered saline to avoid maternal blood and amnioticfluid contamination. Placental DNA was extracted by astandard salting out procedure, modified from Miller etal. [62]. A NanoDrop 1000 spectrophotometer (Thermo-Scientific, USA) was used to assess DNA purity andconcentration.

Candidate single nucleotide polymorphism (SNP)selectionSNPs in 12 innate immune system genes were chosenbased on published findings of an association with anyof chorioamnionitis [48–50], placental inflammation [51,52], or neonatal sepsis/infection [53–55]. The estimatesof the minor allele frequency for the 16 SNPs variedbetween > 1–48% in the general population based on1000 Genomes Project Phase III records [63]. Table 2provides a detailed description of the 16 candidate SNPsinvestigated in the study.Genetic association studies are prone to population

stratification if case and control groups are not matchedby ancestry. Because ancestry was largely unknown inthe study cohort, we also selected 55 ancestry inform-ative marker (AIM) SNPs to genotype in our populationas previously described [56]. These AIM SNPs weredesigned to differentiate between African, European,East Asian, and South Asian ancestries [64–66].

GenotypingAll samples were genotyped using the Sequenom iPlexGold platform at the Génome Québec InnovationCentre (Montréal, Canada). Primary quality control ofthe genotype data comprised of the following steps i)removal of samples with call rate < 90% (n = 1), and ii)removal of SNPs with call rate < 90% (n = 5; 4 AIM SNPs(rs11779571, rs2304925, rs5030240, rs917118) and 1aCA candidate SNP (rs4986790). After primary qualitycontrol, each gene involving multiple SNPs was investi-gated for linkage disequilibrium (LD) to determine if theSNPs were independent of one another. Strong evidencefor LD was observed for two SNP pairs (rs1800795–rs1800796 in IL6 and rs1800896–rs2222202 in IL10)with D’ = 0.99. Haplotype analysis was performed forthese two SNP pairs to investigate whether carriers of aspecific haplotype had increased susceptibility for aCA.The 15 SNPs were also tested for Hardy-Weinberg

Table 1 Identification of variables confounded with acutechorioamnionitis status (pathology)

aCA Non-aCA p-value*

N 72 197

Maternal age (yrs);range (mean)

19.6–44.0 (32.0) 17.0–43.5 (33.0) ns

GA at delivery (wks);range (mean)

20.0–40.0 (28.0) 19.4–41.9 (36.0) 4.32e-15

Birth weight (SD);range (mean)

− 3.10 - 3.05 (− 0.14) −3.13 - 3.23 (0.04) ns

Sex; M/F 38/34 103/94 ns

*p-values are calculated by comparison of aCA cases to non-aCA cases usingWilcoxon-Mann-Whitney rank sum test for continuous variables, Fisher’s exacttest for fetal sex. ns = p > 0.05


Equilibrium (HWE) to detect genotyping error and/orpopulation stratification.

Inferring ancestry of the study populationAfter initial quality control, 50 AIM SNPs were used toinfer ancestry in 269 placental villus DNA samples.Ancestry was described using the top 3 coordinatesderived from a multidimensional scaling (MDS) analysisof AIM SNP genotypes of the placental villus samplesand n = 21,571,000 Genomes Project samples (n = 661African, n = 504 East Asian, n = 504 European, n = 489South Asian) used as ancestry reference populations, asdescribed in Del Gobbo et al. (2018) [56]. Using thismethod, ancestry is assessed as a continuous measure,which is relevant in our genetically heterogeneous studypopulation. Distributions of the three ancestry MDS co-ordinates were compared between aCA and non-aCAgroups to identify any population stratification by ances-try. To select homogenous ancestry groups from our pla-cental chorionic villus samples, an unsupervised clusteringmethod, k-means clustering, with k = 3, was used to clustersamples into three groups of common ancestry.

Publicly available datasetsTo investigate whether candidate SNPs were associatedwith altered DNA methylation, we used our previouslyobtained DNA methylation microarray data that wasavailable for a subset of placental (chorionic villus) sam-ples (n = 67). Some of these (GSE100197 and GSE74738;n = 25) were assessed on the Illumina Infinium Human-Methylation450 BeadChip (450 K array; 485,512 CpG

sites) [67] while the remaining samples (GSE115508;n = 42) were assessed on the Illumina Infinium Human-MethylationEPIC BeadChip (850 k array; 866,895 CpGsites) [68]. Probes for 453,093 CpG sites were present onboth arrays. Probe filtering was performed as describedin Konwar et al. 2018 [58]. To account for type I- type IIprobe bias on the DNA methylation arrays,normalization was performed using ‘preprocessFun-norm’ function in the R minfi package [69]. Principalcomponent analysis (PCA) was used to detect sources ofvariability in the DNA methylation dataset. Known tech-nical variation associated with array type was correctedwith the function “ComBat” in R sva package [70]. DNAmethylation values were reported as β values rangingfrom 0 to 1 (0 = no methylation, 1 = fully methylated)and were used for biological interpretation. However,log-transformed β values, M values, were used for statis-tical analyses as they are less heteroscedastic [71].Further, we took advantage of publicly available data-

sets to determine whether DNA methylation levelscorrelated with gene expression at the associated gene.Our group has previously published gene expressiondata (GSE44711) [72] on a set of 16 chorionic villussamples that were also run on the 450 K array to meas-ure DNA methylation (GSE44667) [72]. Another set of48 matched chorionic villus samples were also utilizedto evaluate correlation between gene expression andDNA methylation levels (GSE98224) [73]. DNA methy-lation data was already normalized and corrected forbatch effects. Log2-transformed expression values wereused for statistical analysis.

Table 2 Information for 16 candidate SNPs in innate immune system genes

Genes Gene name Chromosome SNPs Genomic location

MBL2 Mannose binding lectin 2 10 rs1800450 Exon

TLR2 Toll-like receptor 2 4 rs3804099 Exon

TLR4 Toll-like receptor 4 9 rs1554973 3′ UTR

rs4986790 Exon

rs2149356 Intron

TLR5 Toll-like receptor 5 1 rs5744105 Intron

TLR9 Toll-like receptor 9 3 rs352140 Exon

CD14 Cluster of differentiation 14 5 rs2569190 5′UTR

IL6R Interleukin-6 receptor 1 rs2228144 Exon

IL6 Interleukin-6 7 rs1800795 Promoter

rs1800796 Promoter

IL1B Interleukin-1 beta 2 rs1143643 Intron


rs2222202 Intron


MMP-16 Matrix metalloproteinase-16 8 rs2664349 Intron

*This information is obtained from dbSNP: database from short genetic variations (https://www.ncbi.nlm.nih.gov/snp/)


https://www.ncbi.nlm.nih.gov/snp/

Statistical analysisAll statistical analyses were performed using R version3.4.1. p-values for Table 1 were calculated byWilcoxon-Mann-Whitney rank sum test for continuousvariables and Fisher’s exact test for categorical variables.Kolmogorov-Smirnov (KS) test was used to assess thedifferences in distribution of ancestry MDS coordinatevalues between aCA cases and non-aCA cases. Deviationfrom HWE in controls was assessed using an exact testfor HWE. Statistical tests for differences in allele fre-quencies between aCA cases and non-aCA cases wereconducted with Fisher’s Exact tests. Haplotype analysiswas performed using SNPStats (https://www.snpstats.net/start.htm) [74]. Comparison of DNA methylation levelsbetween genotype groups was carried out using thenon-parametric Kruskal-Wallis test. Spearman’s correla-tions were conducted to determine whether DNAmethylation levels correlated with gene expression at theassociated gene. Power was calculated using the OnlineSample Size estimator, OSSE (http://osse.bii.a-star.edu.sg/index.php).

ResultsCharacterization of population stratification in studypopulationIt is important to determine whether pathology showedevidence of confounding with ancestry, as frequencies ofgenetic variants and the incidence of chorioamnionitis

often vary between different ancestries [17]. There wereno significant differences in the distribution of the threeancestry MDS coordinates between the 72 aCA casesand 197 non-aCA cases (Bonferroni-corrected p > 0.05,Fig. 1). However, we observed that ancestry MDS coord-inate 1, which largely separates European and EastAsians, shows the greatest variability while ancestryMDS coordinate 3, which largely separates the Euro-peans and South East Asians, shows the least variability.Genotype frequencies did not conform to HWE expecta-tions in three SNPs (rs1800795, rs1800796, andrs1554973, p < 0.01). Because rs1800796, rs1800795 andrs1554973 deviated from HWE, we next investigatedwhether the frequencies of these genetic variants wereassociated with ancestry. Significant differences in thedistribution of the two ancestry MDS coordinatesbetween the SNP genotypes was observed (Additionalfile 1: Figure S1), confirming that deviation from HWEat these loci was due to our heterogeneous study cohort.Additionally, both rs1800795 and rs1800796 are in LDand do not conform to HWE, thus it is unlikely thatthese results are due to an artefact of genotyping.

Association of candidate immune SNP allele frequencieswith acute chorioamnionitisTo investigate whether placental (fetal) genetic variationmay lead to an increased susceptibility to developingaCA, we genotyped 16 SNPs within 12 innate immune

Fig. 1 Distribution of ancestry MDS coordinates in the study cohort. The three ancestry MDS coordinates were not significantly different betweenthe aCA cases and non-aCA cases in the study cohort, suggesting pathology was not confounded by ancestry in our study population. p-valueswere calculated by Kolmogorov-Smirnov test


https://www.snpstats.net/start.htm

https://www.snpstats.net/start.htm

http://osse.bii.a-star.edu.sg/index.php

http://osse.bii.a-star.edu.sg/index.php

system genes among our study cohort samples (72 aCA,197 non-aCA). We limited our analysis to SNPs previ-ously implicated in aCA or related phenomenon. Givenour small sample size, we estimated that our study had50% power to detect differences in allele frequenciesassociated with aCA, at a p-value < 0.05, MAF > 1%.Despite this limitation, we found that the minor C alleleat rs1800796 in IL6 was associated with aCA (Fisher’sexact test, p = 0.044). (Additional file 2: Table S1). To ex-plore this further we looked at the genotype distribu-tions for rs1800796 and found that the CC genotype wasassociated with increased risk for aCA (Fisher’s exacttest, p = 0.02, OR = 3.1).The IL6 rs1800796 SNP is known to be strongly corre-

lated with ancestry. Based on 1000 Genomes Projectdata, the C allele is common in individuals of East Asian(70–80%), and rare in individuals of European ancestry(3–5%) (https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/). We also found that the genotype frequen-cies of this SNP varied between different ancestries inour study population (Additional file 1: Figure S1). Wethus sought to explore the association of rs1800796 withaCA within more genetically homogeneous subpopula-tions. K-means clustering grouped samples into threeancestry clusters in our study cohort. The CC genotypewas absent in cluster 1, which was predominantly ofEuropean ancestry (Fig. 2 and Additional file 2: TableS2). Allele frequencies of rs1800796 were significantlyassociated with aCA status only in cluster 3 samples(n = 41) that were largely of East Asian ancestry (Fisher’s

exact test, p = 0.04). Specifically, 8 of the 12 (67%) cases ofaCA in cluster 3 had the “CC” genotype as compared to10 of 29 (34%) non-CA cases. In our study cohort,rs1800796 was in nearly complete LD with rs1800795 (D’= 0.99); however, unlike rs1800796, rs1800795 was poly-morphic in individuals of European ancestry (Additionalfile 2: Table S2) and uninformative in the East Asian clus-ter 3 samples as the GG genotype was completely absent.Although allele frequencies in rs1800795 alone were notassociated with aCA, there was an increased risk of aCAin carriers of the C-C haplotype (p = 0.02).

Association of IL6 rs1800796 genotype with DNAmethylation in IL6A few studies have investigated the relationship betweengenetic variants in IL6 and DNA methylation of theCpGs in the promoter region of IL6 as a mechanismby which genetic variants may modulate disease risk[75–77]. As these studies had been done in blood, wesought to examine whether the IL6 SNP rs1800796was associated with differential DNA methylation inIL6-related CpG sites (n = 8 CpGs) in a subset chori-onic villus samples for which DNA methylationmicroarray data were available (n = 67). Modest (r > 0.5) tostrong (r > 0.7) correlations were observed between βvalues across most of the CpG sites (Additional file 1:Figure S2). DNA methylation was significantly associatedwith rs1800796 genotype at cg01770232 (upstream enhan-cer, p < 1.88e-06), cg02335517 (intronic, p < 6.007e-06),cg07998387 (intronic, p < 4.968e-06), cg13104385 (intronic,

Fig. 2 rs1800796 genotype is associated with ancestry. Based on the ancestry MDS coordinate values, three ancestry clusters were identified by k-means clustering. Of the 22 cases with CC genotype, 11 (50%) were associated with aCA, while 22% (60/246) of the CG/GG genotypes werelinked to aCA. Samples are colored by IL6 rs1800796 genotype


https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/

https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/

p < 0.0003); and cg0526589 (intronic, p < 0.002), wherebyhomozygous C samples showed significant hypermethyla-tion compared to homozygous G samples (Fig. 3). Thesesites have been previously identified as linked to methyla-tion quantitative trait loci (mQTL) in blood [78], meaningCpGs where individual genotypes may result in differentDNA methylation patterns [41]. The CG genotype waspresent in only four samples, therefore we did not includethem in statistical analysis, though as expected, the hetero-zygotes showed intermediate DNA methylation levels(Additional file 1: Figure S3). Further, altered DNA methy-lation at cg01770232, cg7998387, and cg02335517 were alsoassociated with aCA (p < 0.05) (Additional file 1: Figure S4).

Correlation of DNA methylation and gene expression forIL6To evaluate whether DNA methylation is associated withaltered gene transcription activity, we investigated whetherDNA methylation levels of the IL6 CpGs correlated withgene expression at the IL6 locus in chorionic villi. Usingtwo independent publicly available datasets, we observedthat the DNA methylation level at cg01770232 wasnegatively correlated with IL6 expression (GSE44711;GSE44667: r = − 0.67, p < 0.004; GSE98224: r = − 0.56, p <2.937e-05) (Fig. 4). Similar trends were observed for

cg02335517, cg07998387, and cg13104385 (Additional file1: Figure S5).

Association of IL6 expression and DNA methylation withGA, fetal sex or preeclampsia statusUsing the GSE98224 dataset, we observed that IL6 ex-pression in chorionic villus samples was not associatedwith GA or fetal sex (Additional file 1: Figure S6). Al-though previous studies found altered IL6 expression inplacentas from preeclamptic pregnancies, there was nosignificant difference in IL6 expression between PE andnon-PE groups in this dataset (Additional file 1: FigureS6). Using the same dataset, we also did not observe anassociation between DNA methylation at the IL6-relatedCpGs and GA, fetal sex or PE status.

DiscussionIn this study, we sought to validate associations of SNPsin innate immune system genes with aCA status in theplacenta. As the placenta and fetus are genetically identi-cal, risk-conferring genetic variants in innate immunesystem genes may impact inflammation-response path-ways in the placenta and fetus similarly. Additionally,the placenta employs a number of mechanisms to pro-tect the developing fetus from inflammation and/or

Fig. 3 Differentially methylated IL6 CpGs in chorionic villi associated with IL6 rs1800786 genotype. Unadjusted DNA methylation (β) values areplotted on the y-axis w.r.t the CpGs on the x-axis. Carriers of CC genotype showed increased DNA methylation levels compared to carriers of GGgenotype, suggesting DNA methylation levels at these CpG sites are influenced by IL6 genotype. Position of the CpGs are indicated below theboxplots relative to the transcription start site (TSS) (UCSC GRCh37/hg19)


infection [79, 80]. Few studies have reported an associ-ation between genetic variants in inflammatory genes,alterations in immune function and risk for aCA. Themajority of these studies suggest a genetic predispositionof the mother to aCA [48, 49], but the contribution offetal (placental) genetic variants are significantlyunderstudied.In our study sample, we were able to confirm that the

C allele in the IL6 SNP rs1800796 was associated withaCA status (p = 0.04). This allele was linked to increasedDNA methylation, at both an upstream regulatory regionand within the gene body, and associated with decreasedexpression of IL6.Interleukin-6 is a pleiotropic cytokine with a wide

range of biological functions [81]. Primarily, IL6 facili-tates neutrophil recruitment and their subsequent clear-ance from the sites of inflammation [82]. In addition toeliciting innate immune responses, IL6 also regulatesadaptive immunity by influencing proliferation and mat-uration of T cells and B cells [81]. As such, increasedIL6 has been shown to be protective against bacterial in-fection [83]. Therefore, decreased expression observedin the placenta in association with rs1800796 may leadto increased risk for inflammation and aCA.The IL6 SNPs rs1800796 and rs1800795 were previ-

ously associated with increased incidence of aCA anddevelopment of sepsis in children in a study undertakenin Finland [30]. In the cases of rs1800796, the heterozy-gous “CG” genotype was found to be associated withsepsis, and in fact the “CC” genotype was absent fromtheir study population. These same genetic variants havealso been investigated in association with other

inflammatory disorders including chronic periodontitis,systemic onset juvenile chronic arthritis, and distal inter-phalangeal osteoarthritis [84–87]; however, results fromthese studies are conflicting. Inconsistencies across thesestudies may be explained by ancestry differences in thestudy populations as the CC genotype at rs1800796 iscommon in East Asians and rare in individuals ofEuropean ancestry [85, 88, 89]. Though a small sample,we also found no CC genotypes in the placentas of indi-viduals of European ancestry and the C allele was morecommon in individuals of East Asian ancestry (Fig. 2).Further, we found that the allele frequencies ofrs1800796 were associated with aCA status only in indi-viduals of East Asian ancestry, however there was poorpower to detect an effect in Europeans as the CC geno-type was lacking and heterozygotes are expected to haveless of an effect. Similarly, variants at the PGR locus,specifically the PGR SNP rs11224580, that significantlymodulates PGR expression in the ovary has been shownto be common in East Asians compared to individuals ofEuropean and African ancestry [90], but in this case theAsian-specific variant is linked with decreased incidenceof early sPTBs [90]. Because polymorphisms such asrs1800796 (IL6) and rs11224580 (PGR) exhibit extremepopulation-specific allelic variation, these genomic lociare likely to have undergone positive selection that isspecific to Asian populations [90, 91].In addition to genetic variation, circulating levels of IL6

in the serum and amniotic fluid may influence the risk foraCA. Although placental trophoblast cells have the capacityto synthesize IL6 [92, 93], the source of elevated IL6 plasmalevels observed in pregnancy complications such as PE is

Fig. 4 Placental DNA methylation at cg01770232 is associated with IL6 gene expression. DNA methylation at the IL6 CpG site was negativelycorrelated with IL6 log2 transformed gene expression in chorionic villi from a) GSE44711; GSE44667 (r = − 0.67, p = 0.004), and b) GSE98224(r = − 0.56, p = 2.937e-05)


primarily attributed to maternal leukocytes and/or endothe-lial cells [94]. Elevated IL6 levels in the mother may occuras part of a pro-inflammatory response to infection. Incontrast, we found that the placental genotype (rs1800796)associated with aCA is linked with decreased IL6 expres-sion in the placenta. As IL6 mediates a protective immuneresponse against microbial infections [83, 95–98], reducedIL6 expression observed in the placenta may predispose anindividual to infection, by preventing an appropriate innateimmune response to microbes.Although the role of IL6 in modulating innate immune

response has been well-elucidated, molecular mecha-nisms such as DNA methylation underlying the geneticregulation of IL6 transcription have rarely been investi-gated [99]. To our knowledge, this is the first study toshow the role of IL6 genetic variants in modulatingDNA methylation patterns in aCA-affected placentas.We identified that carriers of the rs1800796 C allele hadincreased placental DNA methylation levels at multipleIL6-related CpGs, most significantly at cg01770232,which is located at an upstream enhancer, compared toindividuals with IL6 G allele. Although this CpG hasbeen described as linked to an mQTL in blood, weshowed that this relationship also exists in the placenta.Further, we observed that aCA cases were more methyl-ated than the non-aCA cases at cg01770232, cg07998387,and cg02335517 (Additional file 1: Figure S4).The DNA methylation patterns in the placenta may

imply a primary phenomenon where increased DNAmethylation at cg01770232 is associated with an increasedrisk of developing aCA in individuals carrying the IL6 Callele. Alternatively, some of these subtle DNA methyla-tion changes could be secondary to the disease itself. Al-though we were not able to measure circulating levels ofIL6, using publicly available matched placental DNAmethylation and gene expression data, we observed thatDNA methylation levels at cg01770232 negatively corre-lated with IL6 gene expression. Dandrea et al. (2009) [99]demonstrated that IL6 repression in pancreatic adenocar-cinoma cell lines is facilitated by binding ofmethyl-CpG-binding protein (MeCP2) and H3meK9 tothe methylated CpGs spanning from positions − 666 to −426 relative from the transcription start site of IL6. Inter-estingly, rs1800796 and cg01770232 is located at position572 and 662 respectively, and it is therefore possible thatrs1800796 alters the binding of MeCP2 and H3meK9 tocg01770232, thereby affecting IL6 expression and DNAmethylation, though this has not been tested in placentaltissue. Further, the IL6 upstream region contains several(A/T)> 4 motifs adjacent to the methylated CpGs includingcg01770232, shown to mediate high-affinity MeCP2 bind-ing [100]. Understanding these mechanisms will provideinsights into how genetic variation in IL6 may contributetowards disease pathogenesis in aCA.

Overall, the present study has limitations. Our samplesize was relatively small, especially among the geneticallyhomogenous subpopulations; therefore the findings ofthis study should be evaluated in larger subpopulationsof different ancestries. In particular, the lack of an asso-ciation between the other genetic variants we investi-gated and aCA might be a result of our small samplesize. Although we utilized publicly available datasets toinvestigate functional consequences of the IL6 rs1800796polymorphism, and confirmed that DNA methylationchanges correlated with changes in IL6 gene expression,we could not examine whether IL6 protein levels inmaternal blood would reflect placental DNA methyla-tion and expression. Further, potential confoundingfactors for our study, such as socio-economic status,maternal smoking status, maternal alcohol use, andPPROM status, were not documented for all the casesin our study cohort and thus not accounted for instatistical analyses. Finally, our results only highlightthe biological significance of placental (fetal) geneticvariants in aCA, but this does not exclude the role ofmaternal genetic factors in altering disease risk toaCA, given that maternal genetic effects have beenshown as important contributors to PTB [10].

ConclusionOur findings suggest that a placental (fetal) CC genotypeat the IL6 SNP (rs1800796), which is largely limited toindividuals of East Asian ancestry, is associated withaCA. Placentas with the CC genotype exhibited increasedDNA methylation at multiple CpG sites upstream andwithin IL6, as compared to those of GG genotype. This in-creased DNA methylation was associated with lower ex-pression of IL6 and with aCA status. While overexpressionof IL6 is associated with various inflammatory conditions,this can be a consequence of infection; whereas innate IL6deficiency can lead to impaired immunity against microbialinfection. Taken together, we conclude that IL6 geneticpolymorphisms may influence susceptibility to aCA byaffecting the risk of acute infection. Larger samples sizesare needed to confirm these findings.

Additional files

Additional file 1: Figure S1. Shows the association of SNP genotypes(rs1800795, rs1800796, and rs1554973) with ancestry in study cohort.Figure S2. Shows the correlation of β values across eight IL6-relatedCpGs. Figure S3. Shows differential methylation of IL6-related CpGsbased on IL6 genotype status (rs1800796). Figure S4. Shows altered DNAmethylation at IL6-related CpGs is associated with aCA status. Figure S5.Shows the correlation between placental DNA methylation and geneexpression at IL6 locus. Figure S6. Shows no association of IL6 expressionwith gestational age, fetal sex and preeclampsia status. (DOCX 1205 kb)

Additional file 2: Table S1. Table listing the allele counts and frequenciesof the SNPs used in the statistical analyses. Table S2. Table listing the countsper genotype for IL6 SNPs based on the three clusters. (XLSX 20 kb)


https://doi.org/10.1186/s12881-019-0768-0

https://doi.org/10.1186/s12881-019-0768-0

Abbreviations450 k array: Illumina Infinium HumanMethylation450 BeadChip; 850 karray: Illumina Infinium MethylationEPIC BeadChip; aCA: acutechorioamnionitis; AIM: Ancestry informative marker; CpG: Cytosine-phosphate-guanosine; eQTL: methylation quantitative trait loci;GA: Gestational age; GEO: Gene expression omnibus; HC: Hofbauer cells;IL6: Interleukin-6; KS: Kolmogorov-Smirnov; MDS: Multi-dimensional scaling;mQTL: methylation quantitative trait loci; ns: not significant; PE: Preeclampsia;PTB: Preterm birth; QC: Quality control; SNPs: Single nucleotidepolymorphisms; sPTB: spontaneous preterm birth; TLR: Toll-like receptor

AcknowledgementsThank you to all the participants of this study for generously donating theplacental samples. We also thank K Louie, and Dr. J Schuetz for patientrecruitment; R Jiang and D Hui for placental dissections and DNA extractions;Dr. M Peñaherrera and L Wang for their contributions in sample preparationsfor genotyping. Finally, we are thankful to current and alumnus Robinson labmembers for their insightful discussion and advice.

FundingThis work was supported by an operating grant to WPR from the CanadianInstitutes of Health Research (CIHR) [#49520 to WPR]. WPR receives salarysupport through an investigatorship award from the BC Children’s HospitalResearch Institute.

Availability of data and materialsGenotype/allele data will be available on request. In the present study,publicly available datasets were used to obtain DNA methylation data(GSE100197, GSE74738, GSE115508, GSE44667, GSE98224) and geneexpression data (GSE44711, GSE98224).

Authors’ contributionsCK performed DNA extractions for chorionic villus samples, prepared samplesfor genotyping, participated in study design, conducted statistical analyses,interpreted results, and wrote the draft of the manuscript. GFDG performedthe AIM analysis, assisted in sample preparation for genotyping, statisticalanalyses and results interpretation, and critically revised the manuscript. JTperformed the recruitment and ascertainment of a subset of the studycohort, and critically edited the manuscript. WPR conceived of andsupported the study, contributed to the study design, statistical analyses andresults interpretation, and critically edited the manuscript. All authors readand approved the final manuscript.

Ethics approval and consent to participateEthics approval for this study was obtained from the University of BritishColumbia and the Children’s & Women’s Health Centre of British Columbiaresearch ethics committees (H04–70488). For all samples, only non-identifiable information is presented. Written consent was obtained for thecases in the study when applicable; a subset of cases were de-identifiedbanked samples.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.

Author details1BC Children’s Hospital Research Institute, 950 W 28th Ave, Vancouver, BCV5Z 4H4, Canada. 2Department of Medical Genetics, University of BritishColumbia, Vancouver, BC V6H 3N1, Canada. 3Department of Pathology, BCChildren’s Hospital, Vancouver, BC V6H 3N1, Canada.

Received: 14 November 2018 Accepted: 18 February 2019

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